The antitumor protein Bax is susceptible to microsatellite instability (MSI) mutations that alter its open reading frame by changing Baxs’... Show moreThe antitumor protein Bax is susceptible to microsatellite instability (MSI) mutations that alter its open reading frame by changing Baxs’ microsatellite of eight guanines (G8) to seven guanines (G7). This mutation results in a frameshift that is corrected by alternative splicing, making Bax∆2. Evidence shows that non-MSI mutated full length Bax∆2 (Bax∆2 G8) can be found in tissue. However, the extra guanine in Bax∆2 should result in premature termination of protein synthesis. Therefore, we believe that Bax∆2 is capable of +1 frameshifting to correct the out of frame sequence caused by splicing. The dual luciferase assay system is a useful tool for measuring frameshifting and in this study, we cloned full length Bax∆2 G8 into a dual luciferase vector to analyze frameshifting. Using this method, we found that the full length Bax∆2 G8 sequence has 3.5% frameshifting activity. To further determine whether the frameshifting occurs in or near the G8 microsatellite, we focused on several truncated constructs containing the first three exons. The results from dual luciferase assay showed that frameshifting activity was high in the constructs containing the G8 microsatellite but diminished when the G8 microsatellite region was removed. Surprisingly, constructs containing exon 4 and 5, which are away from the predicted frameshifting region, also showed frameshifting activity. One possibility to explain these results is that mRNA structures, which are critical to frameshifting, could be altered by construct truncation and consequently lead to artificial frameshifting. Thus, using truncated constructs may not be a viable option for testing frameshifting activity. To maintain mRNA integrity, point mutations within the full sequence, could be a better option to identify the frameshifting site. Show less
